Apoptosis signal-regulating kinase 1 controls the proapoptotic function of death-associated protein (Daxx) in the cytoplasm.

Although Daxx (death-associated protein) was first reported to mediate the apoptotic signal from Fas to JNK in the cytoplasm, other data suggested that Daxx is mainly located in the nucleus as a transcriptional regulator. Here, we demonstrated that cellular localization of Daxx could be determined by the relative concentration of a proapoptotic kinase, apoptosis signal-regulating kinase 1 (ASK1) by using immunofluorescence and transcriptional reporter assay. ASK1 sequestered Daxx in the cytoplasm and inhibited the repressive activity of Daxx in transcription. In addition, Daxx was bound to the activated Fas only in the presence of ASK1, accelerating the Fas-mediated apoptosis. These results suggest that Daxx requires ASK1 for its cytoplasmic localization and Fas-mediated signaling. Taken together, we could conclude that ASK1 controls the dual function of Daxx as a transcriptional repressor in the nucleus and as a proapoptotic signal mediator in the cytoplasm.

A cofactor of tRNA synthetase, p43, is secreted to up-regulate proinflammatory genes.

An auxiliary factor of mammalian multi-aminoacyl-tRNA synthetases, p43, is thought to be a precursor of endothelial monocyte-activating polypeptide II (EMAP II) that triggers proinflammation in leukocytes and macrophages. In the present work, however, we have shown that p43 itself is specifically secreted from intact mammalian cells, while EMAP II is released only when the cells are disrupted. Secretion of p43 was also observed when its expression was increased. These results suggest that p43 itself should be a real cytokine secreted by an active mechanism. To determine the cytokine activity and active domain of p43, we investigated tumor necrosis factor (TNF) and interleukin-8 (IL-8) production from human monocytic THP-1 cells treated with various p43 deletion mutants. The full length of p43 showed higher cytokine activity than EMAP II, further supporting p43 as the active cytokine. p43 was also shown to activate MAPKs and NFkappaB, and to induce cytokines and chemokines such as TNF, IL-8, MCP-1, MIP-1alpha, MIP-1beta, MIP-2alpha, IL-1beta, and RANTES. Interestingly, the high level of p43 was observed in the foam cells of atherosclerotic lesions. Therefore, p43 could be a novel mediator of atherosclerosis development as well as other inflammation-related diseases.

An elongation factor-associating domain is inserted into human cysteinyl-tRNA synthetase by alternative splicing.

The amino acid sequence of human cytoplasmic cysteinyl-tRNA synthetase (CRS) was examined by analyzing sequences of genomic and expressed sequence tag fragments. From theses analyses, a few interesting possibilities were suggested for the structure of human CRS. First, different isoforms of CRS may result from alternative splicing. Second, the largest one would comprise 831 amino acids. Third, a new exon was identified encoding an 83 amino acid domain that is homologous to parts of elongation factor-1 subunits as well as other proteins involved in protein synthesis. Northern blot analysis showed three different mRNAs for CRS (of approximately 3.0, 2.7 and 2.0 kb) from human testis while only the 2.7 kb mRNA was commonly detected in other tissues. Expression of the exon 2-containing transcript in testis was confirmed by RT-PCR and northern blotting. CRS containing the exon 2-encoded peptide retained catalytic activity comparable to that lacking this peptide. This peptide was responsible for the specific interaction of CRS with elongation factor-1gamma.

Catalytic peptide of human glutaminyl-tRNA synthetase is essential for its assembly to the aminoacyl-tRNA synthetase complex.

Human glutaminyl-tRNA synthetase (QRS) is one of several mammalian aminoacyl-tRNA synthetases (ARSs) that form a macromolecular protein complex. To understand the mechanism of QRS targeting to the multi-ARS complex, we analyzed both exogenous and endogenous QRSs by immunoprecipitation after overexpression of various Myc-tagged QRS mutants in human embryonic kidney 293 cells. Whereas a deletion mutant containing only the catalytic domain (QRS-C) was targeted to the multi-ARS complex, a mutant QRS containing only the N-terminal appended domain (QRS-N) was not. Deletion mapping showed that the ATP-binding Rossman fold was necessary for targeting of QRS to the multi-ARS complex. Furthermore, exogenous Myc-tagged QRS-C was co-immunoprecipitated with endogenous QRS. Since glutaminylation of tRNA was dramatically increased in cells transfected with the full-length QRS, but not with either QRS-C or QRS-N, both the QRS catalytic domain and the N-terminal appended domain were required for full aminoacylation activity. When QRS-C was overexpressed, arginyl-tRNA synthetase and p43 were released from the multi-ARS complex along with endogenous QRS, suggesting that the N-terminal appendix of QRS is required to keep arginyl-tRNA synthetase and p43 within the complex. Thus, the eukaryote-specific N-terminal appendix of QRS appears to stabilize the association of other components in the multi-ARS complex, whereas the C-terminal catalytic domain is necessary for QRS association with the multi-ARS complex.

Genetic dissection of protein-protein interactions in multi-tRNA synthetase complex.

Cytoplasmic aminoacyl-tRNA synthetases of higher eukaryotes acquired extra peptides in the course of their evolution. It has been thought that these appendices are related to the occurrence of the multiprotein complex consisting of at least eight different tRNA synthetase polypeptides. This complex is believed to be a signature feature of metazoans. In this study, we used multiple sequence alignments to infer the locations of the peptide appendices from human cytoplasmic tRNA synthetases found in the multisynthetase complex. The selected peptide appendices ranged from 22 aa of aspartyl-tRNA synthetase to 267 aa of methionyl-tRNA synthetase. We then made genetic constructions to investigate interactions between all 64 combinations of these peptides that were individually fused to nonsynthetase test proteins. The analyses identified 11 (10 heterologous and 1 homologous) interactions. The six peptide-dependent interactions paralleled what had been detected by crosslinking methods applied to the isolated multisynthetase complex. Thus, small peptide appendices seem to link together different synthetases into a complex. In addition, five interacting pairs that had not been detected previously were suggested from the observed peptide-dependent complexes.

Androstane metabolites bind to and deactivate the nuclear receptor CAR-beta.

The orphan receptor CAR-beta binds DNA as a heterodimer with the retinoid-X receptor and activates gene transcription in a constitutive manner. Here we show that, in contrast to the classical nuclear receptors, the constitutive activity of CAR-beta results from a ligand-independent recruitment of transcriptional co-activators. While searching for potential ligands of CAR-beta, we found that the steroids androstanol and androstenol inhibit the constitutive activity of CAR-beta. This effect is stereospecific: only 3alpha-hydroxy, 5alpha-reduced androstanes are active. These androstanes do not interfere with heterodimerization or DNA binding of CAR-beta; instead, they promote co-activator release from the ligand-binding domain. These androstane ligands are examples of naturally occurring inverse agonists that reverse transcriptional activation by nuclear receptors. CAR-beta (constitutive androstane receptor-beta), therefore, defines an unanticipated steroidal signalling pathway that functions in a manner opposite to that of the conventional nuclear receptor pathways.

Inhibition of estrogen receptor action by the orphan receptor SHP (short heterodimer partner).

SHP (short heterodimer partner) is an unusual orphan receptor that lacks a conventional DNA-binding domain. Previous results have shown that it interacts with several other nuclear hormone receptors, including the retinoid and thyroid hormone receptors, and inhibits their ligand-dependent transcriptional activation. Here we show that SHP also interacts with estrogen receptors and inhibits their function. In mammalian and yeast two-hybrid systems as well as glutathione-S-transferase pull-down assays, SHP interacts specifically with estrogen receptor-alpha (ERalpha) in an agonist-dependent manner. The same assay systems using various deletion mutants of SHP map the interaction domain with ERalpha to the same SHP sequences required for interaction with the nonsteroid hormone receptors such as retinoid X receptor and thyroid hormone receptor. In transient cotransfection assays, SHP inhibits estradiol -dependent activation by ERalpha by about 5-fold. In contrast, SHP interacts with ERbeta independent of ligand and reduces its ability to activate transcription by only 50%. These data suggest that SHP functions to regulate estrogen signaling through a direct interaction with ERalpha.

Novel receptor interaction and repression domains in the orphan receptor SHP.

SHP (short heterodimer partner) is a novel orphan receptor that lacks a conventional DNA binding domain and interacts with other members of the nuclear hormone receptor superfamily. We have characterized the SHP sequences required for interaction with other superfamily members, and have defined an SHP repressor domain. In the mammalian two-hybrid system, a fusion of full-length SHP to the GAL4 DNA binding domain shows 9-cis-retinoic acid-dependent interaction with a VP16-retinoid X receptor alpha (RXR alpha) fusion. By deletion analysis, sequences required for this RXR interaction map to the central portion of SHP (amino acids 92 to 148). The same region is required for interaction with RXR in vitro and in the yeast two-hybrid system, and results from the yeast system suggest that the same SHP sequences are required for interaction with other members of the nuclear hormone receptor superfamily such as thyroid hormone receptor and retinoic acid receptor. In mammalian cells, a GAL4-SHP fusion protein shows about 10-fold-decreased transcriptional activation relative to GAL4 alone, and fusion of SHP to the C terminus of a GAL4-VP16 fusion to generate a triple chimera also results in a strong decrease in transactivation activity. Sequences required for this repressor function were mapped to the C terminus of SHP. This region is distinct from that required for corepressor interaction by other members of the nuclear hormone receptor superfamily, and SHP did not interact with N-CoR in either the yeast or mammalian two-hybrid system. Together, these results identify novel receptor interaction and repressor domains in SHP and suggest two distinct mechanisms for inhibition of receptor signaling pathways by SHP.

Differential transactivation by two isoforms of the orphan nuclear hormone receptor CAR.

We have identified a new murine orphan member of the nuclear hormone receptor superfamily, termed mCAR, that is closely related to the previously described human orphan MB67, referred to here as hCAR. Like hCAR, mCAR expression is highest in liver. In addition to the most abundant mCAR1 isoform, the mCAR gene expresses a truncated mCAR2 variant that is missing the C-terminal portion of the ligand binding/dimerization domain. The mCAR gene has 8 introns, and this mCAR2 variant is generated by a splicing event that skips the 8th exon. mCAR1, like hCAR, binds as a heterodimer with the retinoid X receptor to the retinoic acid response element from the promoter of the retinoic acid receptor beta2 isoform. Consistent with its lack of a critical heterodimerization interface, the mCAR2 variant does not bind this site. Both mCAR1 and hCAR are apparently constitutive transcriptional activators. This activity is dependent on the presence of the conserved C-terminal AF-2 transcriptional activation motif. As expected from its inability to bind DNA, the mCAR2 variant neither transactivates by itself nor inhibits transactivation by hCAR or mCAR1.

Activation of the orphan receptor RIP14 by retinoids.

Retinoids are crucial regulators of a wide variety of processes in both developing and adult animals. These effects are thought to be mediated by the retinoic acid (RA) receptors and the retinoid X receptors (RXRs). We have identified an additional retinoid-activated receptor that is neither a retinoic acid receptors nor an RXR. RXR-interacting protein 14 (RIP14), a recently described orphan member of the nuclear receptor superfamily, can be activated by either all-trans-RA (tRA) or the synthetic retinoid TTNPB [[E]-4-[2-(5, 6, 7, 8-tetrahydro-5, 5, 8, 8-tetramethyl-2-naphthalenyl)propen-1-yl]benzoic acid].RIP14 binds to DNA as a heterodimer with RXR. In the presence of either tRA or TTNPB, the addition of 9-cis-RA or the RXR-specific agonist LG1069 [4-[1-(3, 5, 5, 8, 8-pentamethyl-5, 6, 7, 8-tertrahydro-2-naphthyl)ethenyl]benzoic acid] results in additional activation. Mutations of the ligand-dependent transcriptional activation functions indicate that TTNPB activates the RIP14 component of the RIP14-RXR heterodimer, that 9-cis-RA and LG1069 activate RXR, and that tRA activates via both RIP14 and RXR. Despite the very effective activation of RIP14 by tRA or TTNPB, relatively high concentrations of these compounds are required, and no evidence for direct binding of either compound was obtained using several approaches. These results suggest that RIP14 is the receptor for an as-yet-unidentified retinoid metabolite.

Tap: a novel cellular protein that interacts with tip of herpesvirus saimiri and induces lymphocyte aggregation.

Tip of herpesvirus saimiri associates with Lck and down-regulates Lck-mediated activation. We identified a novel cellular Tip-associated protein (Tap) by a yeast two-hybrid screen. Tap associated with Tip following transient expression in COS-1 cells and stable expression in human Jurkat-T cells. Expression of Tip and Tap in Jurkat-T cells induced dramatic cell aggregation. Aggregation was likely caused by the up-regulated surface expression of adhesion molecules including integrin alpha, L-selectin, ICAM-3, and H-CAM. Furthermore, NF-kappaB transcriptional factor of aggregated cells had approximately 40-fold higher activity than that of parental cells. Thus, Tap is likely to be an important cellular mediator of Tip function in T cell transformation by herpesvirus saimiri.

Two receptor interacting domains in the nuclear hormone receptor corepressor RIP13/N-CoR.

The thyroid hormone receptor (TR) and the retinoic acid receptor (RAR) act as transcriptional repressors when they are not occupied by their cognate ligands. This repressor function is mediated by proteins called corepressors. One of the nuclear hormone receptor corepressors, N-CoR, was originally isolated as a retinoid X receptor-interacting protein called RIP13. We have isolated a new potential variant of RIP13/N-CoR that is missing previously described transcriptional repressor domains but is similar in structure to the related corepressor termed SMRT or TRAC-2. Detailed analysis of the interaction with TR and RAR demonstrates that RIP13/N-CoR contains a new receptor interaction domain, termed ID-II, in addition to the previously described domain, referred to here as ID-I. Both ID-I and ID-II are capable of interacting independently with either TR or RAR, as assessed by the yeast two-hybrid system, by a mammalian two-hybrid system, or by direct in vitro binding. Results with all three approaches confirm that RIP13/N-CoR also interacts with retinoid X receptor, but this interaction is weaker than that with TR or RAR. Together, these results demonstrate that RIP13/N-CoR can interact with several different nuclear hormone receptors via two separate receptor interaction domains. Differences between the interactions observed in the different systems suggest that corepressor function may be modified by additional factors present in various cell types.

An orphan nuclear hormone receptor that lacks a DNA binding domain and heterodimerizes with other receptors.

SHP is an orphan member of the nuclear hormone receptor superfamily that contains the dimerization and ligand-binding domain found in other family members but lacks the conserved DNA binding domain. In the yeast two-hybrid system, SHP interacted with several conventional and orphan members of the receptor superfamily, including retinoid receptors, the thyroid hormone receptor, and the orphan receptor MB67. SHP also interacted directly with these receptors in vitro. In mammalian cells, SHP specifically inhibited transactivation by the superfamily members with which it interacted. These results suggest that SHP functions as a negative regulator of receptor-dependent signaling pathways.

A component of the 26S proteasome binds on orphan member of the nuclear hormone receptor superfamily.

The 26S proteasome complex plays a general role in turnover of both short and long lived proteins by specifically degrading ubiquitinated proteins. Recent evidence suggests that this large protease has more specific functions in a number of important cellular processes, ranging from activation of the transcription factor NFkB and antigen processing to transit through mitosis. We have identified a component of the 26S proteasome that interacts specifically with MB67, an orphan member of the nuclear hormone receptor superfamily. MIP224 (MB67 interacting protein) was isolated using the yeast two hybrid system and is apparently identical to the human 26S proteasome component TBP7. MIP224/TBP7 is one of several proteasomal proteins that share a strongly conserved ATPase domain (CAD) which is also present in a rapidly expanding superfamily of proteins with diverse functions. In yeast, MIP224 interacts specifically with MB67 and another closely related orphan receptor, but does not interact with several other receptor superfamily members tested. In mammalian cells, coexpression of MIP224 inhibits transactivation by MB67. MIP224 also interacts in yeast with other CAD proteins, including MSS1, which is proteasomal, and TRIP1, which is associated with transcriptional activation. This interaction of a proteasomal protein with a transcriptional protein suggests a previously unexpected link between the processes of protein degradation and transcriptional regulation.

Isolation of proteins that interact specifically with the retinoid X receptor: two novel orphan receptors.

We have used a yeast genetic system to isolate cDNAs encoding proteins that specifically interact with the ligand-binding domain of human retinoid X receptor-alpha (RXR alpha). A number encoded portions of two known RXR heterodimer partners, the retinoic acid receptor (RAR) and the peroxisome proliferator activated receptor. Of four additional RXR-interacting proteins (RIPs) selected for further study two, RIP14 and RIP15, are previously unidentified orphan members of the nuclear receptor superfamily. Two others, RIP110 and RIP13, do not show significant similarities to previously reported proteins. RIP110 interacts with LexA-RXR only in yeast cells grown in the presence of the RXR ligand 9-cis-RA, while the interaction of the four receptor superfamily members and RIP13 is unaffected by the presence or absence of 9-cis-RA. RIP110 and RIP13 also interact in yeast with several other members of the receptor superfamily, but RIP14 and RIP15 interact only with RXR. Analysis of larger cDNA clones demonstrates that there are at least two isoforms of RIP14 that differ in the N-terminal (A and B) and hinge (D) domains. Northern blot analysis indicates that RIP14 is expressed specifically in liver and kidney, while RIP15 is expressed in every tissue tested. Both RIP14 and 15 bind as heterodimers with RXR to the RA response element (RARE) from the promoter of the RAR beta 2 isoform (the beta RARE), and RIP14 and RXR heterodimers also bind the ecdysone response element from the Drosophila heat shock protein 27 promoter. Both heterodimers also bind to several synthetic RAREs and other elements. In cotransfections, neither RIP14 nor RIP15 trans-activates a reporter containing multiple copies of the beta RARE under any of a variety of conditions, suggesting that their activities are dependent on the binding of as yet unidentified specific ligands or on activation by other processes.

Membrane topology model of Escherichia coli alpha-ketoglutarate permease by phoA fusion analysis.

Escherichia coli alpha-ketoglutarate permease (KgtP) is a 432-amino-acid protein that symports alpha-ketoglutarate and protons. KgtP was predicted to contain 12 membrane-spanning domains on the basis of a calculated hydropathy profile. The membrane topology model of KgtP was analyzed by using kgtP-phoA gene fusions and measuring alkaline phosphatase activities in cells expressing the chimeric proteins. Comparisons of the phosphatase activity levels and the locations of the KgtP-PhoA junctions are consistent with the predicted membrane topology model of KgtP.

Site-directed mutants of Escherichia coli alpha-ketoglutarate permease (KgtP).

To investigate an active site(s) in the Escherichia coli alpha-ketoglutarate premease, 11 point mutants were made in the corresponding structural gene, kgtP, by oligonucleotide-directed mutagenesis and the polymerase chain reaction. On the basis of sequences conserved in KgtP and related members of a transporter superfamily [Henderson P. J. F., & Maiden, M. C. (1990) Philos. Trans. R. Soc. London B 326, 391], Arg76 was replaced with Ala, Asp, or Lys; Asp88 with Asn or Glu; His90 with Ala; Arg92 with Ala or Lys; and Arg198 with Ala, Asp, or Lys. Mutant proteins expressed using the T7 polymerase system were in each case shown to be membrane-associated. However, they differed in transport activity. Mutants H90A and R198K had activities similar to that of wild type, and R76K and R198A retained 10-60% of the wild-type activity. In all other mutants, alpha-ketoglutarate transport was abolished. The results suggest that Arg92, which is highly conserved among other members of the transporter superfamily, is necessary for activity and also that Asp88 is critical for function, as observed for the tetracycline transporter. These data show further that a positive charge is essential at position 76 and is also important, but not absolutely required, at position 198 for alpha-ketoglutarate transport. Unlike lacY permease which was inactivated by deleting the last helix [McKenna, E., Hardy, D., Pastore, J. C., & Kaback, H. R. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 2969], a KgtP truncation mutant missing the last putative membrane-spanning region was relatively stable and also retained 10-50% of the wild-type level of alpha-ketoglutarate transport activity.

Escherichia coli alpha-ketoglutarate permease is a constitutively expressed proton symporter.

Escherichia coli kgtP which maps at 56.5 min codes for alpha-ketoglutarate permease (KgtP). This protein, expressed from the cloned gene using the T7 polymerase system and [35S]methionine labeling, fractionated with cell membranes. Right-side-out (RSO) membrane vesicles prepared from a kgtP negative mutant strain did not transport alpha-ketoglutarate, but RSO vesicles from the same strain expressing KgtP from a transforming plasmid transported alpha-ketoglutarate effectively as measured by uptake of the 14C-labeled substrate. E. coli JC7623 strain grown in M9 minimal medium with glucose, glycerol, or alpha-ketoglutarate as carbon source contained a 1.3-kilobase RNA which hybridized to nick-translated kgtP probe. In addition, strain MC1061 cultures grown under these same conditions were all capable of transporting alpha-ketoglutarate, demonstrating that KgtP is constitutively expressed. The Km and Vmax of KgtP assayed in strain MC1061 vesicles were 13-46 microM and 8 nmol/min/mg protein, respectively. Uncouplers that permeabilized the membrane to protons inhibited alpha-ketoglutarate transport into energized vesicles, and the addition of alpha-ketoglutarate to vesicle suspensions under non-energized conditions resulted in an increase in pH. These results indicate that KgtP is an alpha-ketoglutarate-proton symporter.

Escherichia coli kgtP encodes an alpha-ketoglutarate transporter.

The witA gene located between pss and rrnG on the Escherichia coli chromosome encodes a 432-amino acid protein. It is homologous to a human hepatoma glucose transporter and to E. coli membrane proteins that transport citrate (CitA), arabinose (AraE), and xylose (XylE), and, like these carrier proteins, WitA also contains 12 highly hydrophobic putative membrane-spanning regions. Gene disruption mutants constructed in two E. coli strains grew slowly or not at all, depending on genetic background, in M9 minimal medium containing alpha-ketoglutarate. Growth on alpha-ketoglutarate and uptake of alpha-[14C]ketoglutarate were restored by transformation with plasmids containing witA. These complementation studies indicate that WitA is an alpha-ketoglutarate transporter and should be renamed kgtP(alpha-ketoglutarate permease).